(1) A cytogenetic assay for G2 phase DNA repair using blood lymphocytes was developed. with this assay, Down syndrome (DS), like other cancer- prone genetic disorders, was shown to have a G2 phase DNA repair deficiency. (2) Of 69 individuals assayed who had no known cancer-prone genetic disease, 51 had efficient and 18 deficient repair. Individuals with the deficient G2 response had first- and second-degree relatives with a 3.6- and 2.2-fold higher mean frequency of cancer, respectively. The results suggest that this deficient response to G2 phase X- irradiation is associated with a high risk of cancer. (3) A cytogenetic assay for capacity of cells to incise DNA at damaged sites after G2 X- irradiation indicated negligible incision activity in xeroderma pigmentosum (XP) A and D cells, a higher level in XP-C and a deficiency in neurologic diseases, DS and Alzheimer's disease. Results suggest that this deficiency may be associated with neurodegeneration. (4) Human skin keratinocytes after malignant transformation by ras oncogene showed a DNA repair deficiency. Control non-tumorigenic cells and clones transfected with ras oncogene that grew as benign tumors and expressed the mutated p21 protein were repair-efficient. Only those transfected clones that had acquired the DNA repair deficiency grew as carcinomas in nude mice. Acquisition of deficient DNA repair after ras oncogene transfection may provide the genetic instability required for progression from benign to malignant state. (5) Skin fibroblasts from 6 family members with Li- Fraumeni syndrome showed the G2 phase repair deficiency in contrast to 3 normal controls. All 6 members of the family had a radioresistant phenotype as determined by cell killing. Fibroblasts from a radioresistant family member showed the same extent of chromatid damage directly after G2 phase X-irradiation as those from the radiosensitive control spouse. It is concluded, therefore, that radiosensitivity, as determined by cell killing in asynchronous cell populations is unrelated to chromosomal sensitivity to G2 phase X-irradiation. However, the persistence of a high frequency of chromatid breaks and gaps after G2 phase X-irradiation, a manifestation of deficient DNA repair, is associated with cancer-proneness in this family.